Advantages of a new Taq DNA polymerase in multiplex PCR and time-release PCR.
Extensive diagnostic and scientific investigations are often restricted by limited availability of material. Therefore, methods like multiplex PCR strategies are needed to conserve as much sample as possible. Unfortunately, the establishment of such procedures poses several difficulties. Here we describe the advantages of a new enzyme, AmpliTaq Gold DNA Polymerase, in multiplex and time-release PCR. The application of this thermostable recombinant Taq DNA polymerase allows the specific amplification of DNA/cDNA targets with very high sensitivity. With our protocol, the specific amplification of 13 different cDNAs of cytokines and cytokine receptors can be realized in three multiplex PCRs (IL-2R alpha, IL-2/15R beta, gamma c-chain, IL-4 and IL-4R alpha; IL-10, IL-15 and IL-15R alpha; and IL-2, IFN gamma, IL-7, IL-7R alpha and IL-9R alpha). The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected. No IL-9 cDNA can be detected in peripheral blood mononuclear cells (PBMC) in the absence of any stimulation, thus it was difficult to amplify this target with routine PCR protocols. Here we demonstrate the reliable and reproducible amplification of IL-9 cDNA in the Hodgkin's lymphoma cell line KM-H2, in PBMC and in stimulated PBMC. Results with AmpliTaq Gold DNA Polymerase were more sensitive and specific compared with AmpliTaq DNA Polymerase, with and without manual hot-start procedure.
Kebelmann-Betzing
C
C
Virchow Medical Center Charité, Humboldt-University, Berlin, Germany.
Seeger
K
K
Dragon
S
S
Schmitt
G
G
Möricke
A
A
Schild
T A
TA
Henze
G
G
Beyermann
B
B
D018076:DNA, Complementary / Q000032:analysis; D006801:Humans; D016906:Interleukin-9 / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism*
D018076:DNA, Complementary; D016906:Interleukin-9; D019914:Taq Polymerase